Novel pyridazine compound and use thereof

ABSTRACT

A compound of formula (1) and its salts and solvates are provided for the treatment or prophylaxis of hepatitis C virus infections 
     
       
         
         
             
             
         
       
     
     Methods of making and formulating compound (1) are provided.

BACKGROUND OF THE INVENTION

The hepatitis C virus is an enveloped, single-stranded, positive senseRNA virus in the family Flaviviridae. HCV mainly replicates withinhepatocytes in the liver. Circulating HCV particles bind to receptors onthe surfaces of hepatocytes and subsequently enter the cells. Onceinside the hepatocyte, HCV utilizes the intracellular machinerynecessary to accomplish its own replication. Lindenbach, B. Nature436(7053):932-8 (2005). The HCV genome is translated to produce a singleprotein of around 3011 amino acids. This “polyprotein” is thenproteolytically processed by viral and cellular proteases to producethree structural (virion-associated) and seven nonstructural (NS)proteins.

HCV encodes two proteases, the NS2 cysteine autoprotease and the NS3-4Aserine protease. The NS proteins then recruit the viral genome into anRNA replication complex, which is associated with rearranged cytoplasmicmembranes. RNA replication takes places via the viral RNA-dependent RNApolymerase of NS5B, which produces a negative-strand RNA intermediate.The negative strand RNA then serves as a template for the production ofnew positive-strand viral genomes. Nascent genomes can then betranslated, further replicated, or packaged within new virus particles.New virus particles presumably bud into the secretory pathway and arereleased at the cell surface.

HCV has a high rate of replication with approximately one trillionparticles produced each day in an infected individual. Due to lack ofproofreading by the HCV RNA polymerase, HCV also has an exceptionallyhigh mutation rate, a factor that may help it elude the host's immuneresponse.

Based on genetic differences between HCV isolates, the hepatitis C virusspecies is classified into six genotypes (1-6) with several subtypeswithin each genotype. Subtypes are further broken down into quasispeciesbased on their genetic diversity. The preponderance and distribution ofHCV genotypes varies globally. For example, in North America genotype 1apredominates followed by 1b, 2a, 2b, and 3a. In Europe genotype 1b ispredominant followed by 2a, 2b, 2c, and 3a. Genotypes 4 and 5 are foundalmost exclusively in Africa. Genotype is clinically important indetermining potential response to interferon-based therapy and therequired duration of such therapy. Genotypes 1 and 4 are less responsiveto interferon-based treatment than are the other genotypes (2, 3, 5 and6). Duration of standard interferon-based therapy for genotypes 1 and 4is 48 weeks, whereas treatment for genotypes 2 and 3 is completed in 24weeks.

The World Health Organization estimates that world-wide 170-200 millionpeople (3% of the world's population) are chronically infected with HCV.Approximately 75% of these individuals are chronically infected withdetectable HCV RNA in their plasma. These chronic carriers are at riskof developing cirrhosis and/or liver cancer. In studies with a 7-16years follow-up, 7-16% of the patients developed cirrhosis, 0.7-1.3%developed hepatocellular carcinoma and 1.3-3.7% died of liver-relateddisease.

The only treatment option available today is the use of interferon α-2(or its pegylated form) either alone or combined with ribavirin.However, sustained response is only observed in about 40% of thepatients and treatment is associated with serious adverse effects. Thereis thus an urgent need for potent and selective inhibitors of HCV.

Relevant disclosures include U.S. Pat. Nos. 4,914,108; 4,988,707;4,990,518; 5,137,896; 5,208,242; 5,227,384; 5,302,601; 5,374,638;5,405,964; 5,438,063; 5,486,525; 6,479,508; and U.S. Patent PublicationNo. US2003/0108862 A1, Canadian Patent No. 2423800 A1, German PatentNos. 4211474 A1, 4236026, 4309969, 4318813, European Patent Nos. EP 0138 552 A2, EP 0 706 795 A2, EP 1 132 381 A1, Great Britain Patent No.2158440 A, PCT Patent Publication Nos. WO 00/20416, WO 00/39127, WO00/40583, WO 03/007945 A1, WO 03/010140 A2, WO 03/010141 A2, WO93/02080, WO 93/14072, WO 96/11192, WO 96/12703, WO 99/27929,PCT-US2004/43112, PCT-BE2003/000117, PCT-US2005/26606, Akamatsu, et al.,“New Efficient Route for Solid-Phase Synthesis of BenzimidazoleDerivatives”, 4:475-483, J. COMB. CHEM., 2002, Baginski S G et al.,Proc. Natl. Acad. Sci. U.S.A. 2000 Jul. 5;97(14):7981-6). Cleve et al.,“Derivate des Imidazo[4.5-b]- and lmidazo[4.5-c]pyridins”, 747:158-171,JUSTUS LIEBIGS ANNALEN DER CHEMICA, 1971, Kiyama, et al., “Synthesis andEvaluation of Novel Nonpeptide Angiotensin II Receptor Antagonists:Imidazo[4,5-c]pyridine Derivatives with an Aromatic Substituent”,43(3):450-60, CHEM PHARM BULL, 1995, Mederski et al., “Synthesis andStructural Assignment of Some N-substituted ImidazopyridineDerivatives”, 48(48):10549-58, TETRAHEDRON, 1992, Yutilov et al.,23(1):56-9, KHIMIKO-FARMATSEVTICHESKII ZHURNAL, 1989. In addition, seeWO 05/063744.

A need exists for compounds having desired anti-HCV therapeutic and/orprophylactic attributes, including high potency, selectivity and oralbioavailability (suitable for administration once or twice a day), lowtoxicity (including acceptable performance in the hERG patch clampassay, absence of pulmonary permeability edema and no effect on QTinterval), minimal or no metabolic activation/glutathione adductformation, no evidence of genotoxicity, low metabolic turnover and lowplasma clearance, wide-spectrum efficacy against HCV genotypes(especially 1a and 1b, 2 , 3 and 4), efficacy against HCV resistancemutations (limited overlap in resistance profiles with othernon-nucleoside NS5B inhibitors in clinical trials), and compatibilitywith other HCV therapeutics such as interferon and ribavirin. The safetyprofile should permit chronic dosing for periods of at least I year.

SUMMARY OF THE INVENTION

In accordance with achieving the foregoing objects of this invention, acompound is provided having formula (1)

together with its salts and solvates. IUPAC:5-({6-[2,4-bis(trifluoromethyl)phenyl]pyridazin-3-yl}methyl)-2-(2-fluorophenyl)-5H-imidazo[4,5-c]pyridine.CAS: 5H-imidazo[4,5-c]pyridine,5-[[6-[2,4-bis(trifluorornethyl)phenyl]pyridazin-3-yl]methyl]-2-(2-fluorophenyl).

Compound (1) is useful in a method for therapy or prophylaxis of HCVinfection comprising administering to a subject a therapeutic orprophylactic dose of compound (1). Another embodiment comprises the useof compound (1) for the manufacture of a medicament for the preventionor treatment of a HCV infection in a mammal (more specifically a human).

Another embodiment of this invention is a method for making a compoundof formula (1)

comprising (a) reacting5-[6-chloro-pyridazin-3-ylmethyl]-2-(2-fluoro-phenyl)-5H-imidazo[4,5-c]pyridinewith 2,4-bis(trifluoromethyl)phenylboronic acid in the presence of asolvent having the structure R¹OR²O(R⁴O)_(a)R³ wherein each of R¹, R²,R³ and R⁴ are independently selected from is C1-C6 alkyl and a is 0 or1, and (b) recovering compound (1).

In another embodiment for the manufacture of compound (1), a method isprovided comprising providing the intermediate (2)

coupling 2,4-bis(trifluoromethyl)phenylboronic acid to3-chloro-6-methylpyridazine to produce compound (2a)

treating compound (2a) with a chlorinating agent to produce thealkylating agent (3)

and using alkylating agent (3) to alkyl ate intermediate (2) under basicconditions to yield compound (1)

The alkylating agent (3) is new and is part of this invention, as is thesame compound having methyl substitution rather than chloromethyl, orbromo, fluoro or iodo in place of chloro.

Another embodiment of this invention relates to pharmaceuticalcompositions of the formula (1) compound comprising at least onepharmaceutically acceptable excipient. In one embodiment the compound offormula (1) is formulated with an organic acid and optionally formulatedinto a pharmaceutic dosage form such as a capsule. In anotherembodiment, compound (1) is micronized and formulated as a suspension.

Compound (1) or the pharmaceutical compositions of this invention areemployed in the treatment or prophylaxis of hepatitis C.

FIGURES

FIG. 1 depicts an X-ray powder diffraction pattern obtained for crystalform compound (1) reference standard obtained by the method of example1b.

FIG. 2 is an X-ray powder diffraction pattern obtained for amorphousform compound (1) Research Lot 6, obtained by the method of Example 1a.

FIG. 3 illustrates a DSC thermogram obtained for crystal form compound(1) reference standard, 1° C./min scan, obtained by the method ofexample 1b.

FIG. 4 shows a DSC thermogram obtained for amorphous form compound (1)Research Lot 6, 5° C./min scan, obtained by the method of example 1a.

DETAILED DESCRIPTION OF THE INVENTION

The therapeutic compound of this invention is administered to a subjectmammal (including a human) by any means well known in the art, i.e.orally, intranasally, subcutaneously, intramuscularly, intradermally,intravenously, intra-arterially, parenterally or by catheterization in atherapeutically effective amount, i.e., an HCV-inhibiting amount or anHCV-replication inhibiting amount. This amount is believed to be anamount that ensures a plasma level of about 100 nM, 3 times the proteinadjusted EC90. This ordinarily is expected to be achieved by oraladministration of about 0.5—about 5 mg/kg, typically about 0.7 to 2.2mg/kg, most ordinarily about 1.2 mg/kg bodyweight for humans.

The optimal dosage of the compound of this invention will depend uponmany factors known to the artisan, including bioavailability of thecompound in a given formulation, the metabolism and distribution of thecompound in the subject, the fasted or fed state of the subject,selection of carriers and excipients in the formulation, and otherfactors. Proper dosing typically is determined in the preclinical andclinical settings, and is well within the skill of the ordinary artisan.The therapeutically effective amount of the compound of this inventionoptionally is divided into several sub-units per day or is administereddaily or in more than one day intervals, depending upon the nature ofthe infection, the patient's general condition and the formulation ofthe compound of this invention. Generally, the compound is administeredtwice daily.

The compound of this invention is employed in concert with other agentseffective against HCV infections. They optionally are administeredseparately in a course of therapy, or are combined with compound (1) ina unitary dosage form such as tablet, iv solution or capsule. Such otheragents include, for instance, interferon-alpha, ribavirin, and/orcompounds falling within the disclosures of EP1162196, WO 03/010141, WO03/007945, WO 00/204425 and/or WO 03/010140 (and other filings withintheir patent families). Other agents for administration in a course oftherapy with the compound of this invention include compounds now inclinical trials, in particular HCV protease inhibitors such as VX-950(Vertex Pharmaceuticals), SCH 5030347 (Schering Plough) and BILN-2061(Boehringer Ingelheim), nucleoside HCV inhibitors such as NM283, NM107(both Idenix/Novartis) and R1626 (Hoffmann-LaRoche), and non-nucleosideHCV inhibitors including HCV-086 and -796 (both ViroPharma/Wyeth).Supplementary antiviral agents are used in conventional amounts,although if the efficacy of the compound of this invention and thesupplementary compound are additive then the amounts of each activeagent optionally are commensurately reduced, and more so if the agentsact synergistically. In general, however, the agents are used in theirordinary active amounts in unitary combination compositions.

Co-administered agents generally are formulated into unitarycompositions with the compound of this invention so long as they arechemically compatible and are intended to be administered by the sameroute. If not, then they optionally are provided in the form of amedical kit or package containing the two agents in separaterepositories or compartments.

The compound of this invention is provided as the free base or as asalt. Salts typically are prepared by acid addition of organic and/orinorganic acids to the free base. Examples include (1) inorganic acidssuch as hydrohalogen acids, e.g. hydrochloric or hydrobromic acid,sulfuric acid, nitric acid, phosphoric acid and sulfamic acids; or (2)organic acids such as acetic, propanoic, hydroxyacetic, benzoic,2-hydroxypropanoic, 2-oxopropanoic, lactic, fumaric, tartaric, pyruvic,maleic, malonic, malic, salicylic (e.g. 2-hydroxybenzoic),p-aminosalicylic, isethionic, lactobionic, succinic, oxalic and citricacids; organic sulfonic acids, such as methanesulfonic, ethanesulfonic,benzenesulfonic, p-toluenesulfonic, C1-C6 alkylsulfonic,benzenesulfonic, p-toluenesulfonic, and cyclohexanesulfamic acids.Typical salts are the chloride, sulfate, bisulfate, mesylate, besylate,esylate, phosphate, oxalate, maleate, succinate, citrate, malonate,and/or fumarate. Also included within the scope of this invention arethe salts of the compound of this invention with one or more aminoacids, typically naturally-ocurring amino acids such as one of the aminoacids found in proteins. The acidic counterion desirably isphysiologically innocuous and non-toxic or otherwise pharmaceuticallyacceptable, unless the salt is being used as an intermediate inpreparation of the compounds whereupon toxicity is not relevant.Ordinarily, compound (1) will be administered as the free base, butsuitable salts include mesylate (methanesulfonic acid) and HCl.

The compound of this invention includes the solvates formed with thecompound of this invention or their salts, such as for example hydrates,alcoholates and the like.

The pharmaceutical compound of this invention optionally is formulatedwith conventional pharmaceutical carriers and excipients, which will beselected in accord with ordinary practice. Tablets will containexcipients, glidants, fillers, binders and the like. Aqueousformulations are prepared in sterile form, and when intended fordelivery by other than oral administration generally will be isotonic.Formulations optionally contain excipients such as those set forth inthe “Handbook of Pharmaceutical Excipients” (2005) and include ascorbicacid and other antioxidants, chelating agents such as EDTA,carbohydrates such as dextrin, hydroxyalkylcellulose,hydroxyalkylmethylcellulose and/or organic acids such as oleic acid orstearic acid.

The term “pharmaceutically acceptable carrier” as used herein means anymaterial or substance formulated with the active ingredient in order tofacilitate its preparation and/or its application or dissemination tothe site to be treated. Suitable pharmaceutical carriers for use in thecompositions of this invention are well known to those skilled in theart. They include additives such as wetting agents, dispersing agents,adhesives, emulsifying agents, solvents, glidants, coatings,antibacterial and antifungal agents (for example phenol, sorbic acid,chlorobutanol), and isotonic agents (such as sugars or sodium chloride),provided that the same are consistent with pharmaceutical practice, i.e.they are not toxic to mammals.

The pharmaceutical compositions of the present invention are prepared inany known manner, for instance by homogeneously mixing, coating and/orgrinding the active ingredients in a one-step or multi-step procedure,with the selected carrier material and, where appropriate, otheradditives such as surface-active agents. Compositions containing thecompound of this invention formulated into microspheres (usually havinga diameter of about 1 to 10 gm) are useful as controlled or sustainedrelease formulations.

In one optional formulation, compound (1) is comminuted to a finelydivided form, typically to an average particle size at any point withinthe range of about 1-20 microns. The product of example lb iscrystalline needles and exhibits a range of crystal sizes, typicallyabout 25-40 microns. This optionally is micronized in a Jet mill-00 atabout 60-80 psi to obtain particles of about 3-4 microns and havingsurface area of about 7-8 square meters/g. However, the starting crystalsizes will vary from lot to lot and the degree of micronization is amatter of choice. Accordingly, micronized compound (1) is simply definedas crystal or amorphous compound (1) that has been subject to amicronization process such as the exemplary one described here. Neitherthe size nor surface area of the resulting particles is critical. Themicronized compound (1) is suspended in aqueous solution, optionallyaided by a suspending agent, emulsifiers and/or surfactant as furtherdescribed below.

Typically, the pharmaceutical formulation is a solubilized form ofcompound (1) where compound (1) is dissolved in an appropriate solventor solubilizing agent, or combinations thereof. Compound (1) solubilizedin organic solvent is useful as an intermediate for the preparation ofcrystalline compound (1), but typically it is solubilized in apharmaceutically acceptable excipient for administration therapeuticallyor prophylactically.

Suitable solutions of compound (1) for pharmaceutical preparationsinclude water together with various organic acids (typically C4-C24)usually fatty acids like capric, oleic, lauric, capric, palmitic and/ormyristic acid. The fatty acids are optionally saturated or unsaturated,or mixtures thereof. In addition, polyethylene glycols (PEGs) and/orshort, medium, or long chain mono, di, or triglycerides are employedsupplementary to, or in place of, the organic acids. Pegylated short,medium or long chain fatty acids optionally also are used in the samefashion.

The most common organic acids are the carboxylic acids whose acidity isassociated with the carboxyl group —COOH. Sulfonic acids, containing thegroup OSO₃H, are relatively stronger acids for use herein. In general,the acid desirably contains a lipophilic domain. Mono- or di-carboxylicacids are suitable.

Suitable surface-active agents optionally are used with any of theformulations of this invention (any one or more of the following agents,typically any one of them). Such agents also are known as emulgents oremulsifiers, and are useful in the pharmaceutical compositions of thepresent invention. They are non-ionic, cationic and/or anionic materialshaving suitable emulsifying, dispersing and/or wetting properties.Suitable anionic surfactants include both water-soluble soaps andwater-soluble synthetic surface-active agents. Suitable soaps arealkaline or alkaline-earth metal salts, unsubstituted or substitutedammonium salts of higher fatty acids (C₁₀-C₂₂), e.g. the sodium orpotassium salts of oleic or stearic acid, or of natural fatty acidmixtures obtainable from coconut oil or tallow oil. Syntheticsurfactants include sodium or calcium salts of polyacrylic acids; fattysulphonates and sulphates; sulphonated benzimidazole derivatives andalkylarylsulphonates. Fatty sulphonates or sulphates are usually in theform of alkaline or alkaline-earth metal salts, unsubstituted ammoniumsalts or ammonium salts substituted with an alkyl or acyl radical havingfrom 8 to 22 carbon atoms, e.g. the sodium or calcium salt oflignosulphonic acid or dodecylsulphonic acid or a mixture of fattyalcohol sulphates obtained from natural fatty acids, alkaline oralkaline-earth metal salts of sulphuric or sulphonic acid esters (suchas sodium lauryl sulphate) and sulphonic acids of fatty alcohol/ethyleneoxide adducts. Suitable sulphonated benzimidazole derivatives preferablycontain 8 to 22 carbon atoms. Examples of alkylarylsulphonates are thesodium, calcium or alcoholamine salts of dodecylbenzene sulphonic acidor dibutyl-naphthalenesulphonic acid or a naphthalene-sulphonicacid/formaldehyde condensation product. Also suitable are thecorresponding phosphates, e.g. salts of phosphoric acid ester and anadduct of p-nonylphenol with ethylene and/or propylene oxide, orphospholipids. Suitable phospholipids for this purpose are the natural(originating from animal or plant cells) or synthetic phospholipids ofthe cephalin or lecithin type such as e.g. phosphatidylethanolamine,phosphatidylserine, phosphatidylglycerine, lysolecithin, cardiolipin,dioctanylphosphatidyl-choline, dipalmitoylphoshatidyl-choline and theirmixtures. Aqueous emulsions with such agents are within the scope ofthis invention.

Suitable non-ionic surfactants include polyethoxylated andpolypropoxylated derivatives of alkylphenols, fatty alcohols, fattyacids, aliphatic amines or amides containing at least 12 carbon atoms inthe molecule, alkylarenesulphonates and dialkylsulphosuccinates, such aspolyglycol ether derivatives of aliphatic and cycloaliphatic alcohols,saturated and unsaturated fatty acids and alkylphenols, said derivativespreferably containing 3 to 10 glycol ether groups and 8 to 20 carbonatoms in the (aliphatic) hydrocarbon moiety and 6 to 18 carbon atoms inthe alkyl moiety of the alkylphenol. Further suitable non-ionicsurfactants are water-soluble adducts of polyethylene oxide withpoylypropylene glycol, ethylenediaminopolypropylene glycol containing 1to 10 carbon atoms in the alkyl chain, which adducts contain 20 to 250ethyleneglycol ether groups and/or 10 to 100 propyleneglycol ethergroups. Such compounds usually contain from 1 to 5 ethyleneglycol unitsper propyleneglycol unit. Representative examples of non-ionicsurfactants are nonylphenol-polyethoxyethanol, castor oil polyglycolicethers, polypropylene/polyethylene oxide adducts,tributylphenoxypolyethoxyethanol, polyethyleneglycol andoctylphenoxypolyethoxyethanol. Fatty acid esters of polyethylenesorbitan (such as polyoxyethylene sorbitan trioleate), glycerol,sorbitan, sucrose and pentaerythritol are also suitable non-ionicsurfactants.

Suitable cationic surfactants include quaternary ammonium salts,particularly halides, having 4 hydrocarbon radicals optionallysubstituted with halo, phenyl, substituted phenyl or hydroxy; forinstance quaternary ammonium salts containing as N-substituent at leastone C8-C22 alkyl radical (e.g. cetyl, lauryl, palmityl, myristyl andoleyl) and, as further substituents, unsubstituted or halogenated loweralkyl, benzyl and/or hydroxy-lower alkyl radicals.

A more detailed description of surface-active agents suitable for thispurpose is found in “McCutcheon's Detergents and Emulsifiers Annual” (MCPublishing Crop., Ridgewood, N.J., 1981), “Tensid-Taschenbucw”, 2nd ed.(Hanser Verlag, Vienna, 1981) and “Encyclopaedia of Surfactants,”(Chemical Publishing Co., New York, 1981).

The compound of this invention is administered by any route appropriateto the condition to be treated, such as oral, rectal, nasal, topical(including ocular, buccal and sublingual), vaginal and parenteral(including subcutaneous, intramuscular, intravenous, intradermal,intrathecal and epidural). The preferred route of administration mayvary with for example the condition of the recipient, but is generallyoral.

Formulations of the compound of this invention for oral administrationusually are presented as discrete units such as capsules, cachets ortablets each containing a predetermined amount of the active ingredient;as a powder or granular form; as a solution or suspension in an aqueousliquid or a non-aqueous liquid; or as an oil-in-water liquid emulsion ora water-in-oil liquid emulsion. The compound of this inventionoptionally is presented as a bolus, electuary or paste.

A tablet is made by compression or molding, optionally with one or moreaccessory ingredients. Compressed tablets are prepared by compressing ina suitable machine the compound of the invention in a free-flowing formsuch as a powder or granules, optionally mixed with a binder, lubricant,inert diluent, preservative, surface active and/or dispersing agent.Molded tablets typically are made by molding in a suitable machine amixture of the powdered compound moistened with an inert liquid diluent.The tablets may optionally be coated or scored and may be formulated soas to provide slow or controlled release of the active ingredienttherein.

The formulations are optionally applied as a topical ointment or creamcontaining the active ingredient(s) in an amount of, for example, 0.075to 20% w/w (including active ingredient(s) in a range between 0.1% and20% in increments of 0.1% w/w such as 0.6% w/w, 0.7% w/w, etc),preferably 0.2 to 15% w/w and most preferably 0.5 to 10% w/w. Whenformulated in an ointment, the compound is employed with a paraffinic ora water-miscible ointment base. Alternatively, the compound isformulated in a cream with an oil-in-water cream base. If desired, theaqueous phase of the cream base may include, for example, at least 30%w/w of a polyhydric alcohol, i.e. an alcohol having two or more hydroxylgroups such as propylene glycol, butane 1,3-diol, mannitol, sorbitol,glycerol and polyethylene glycol (including PEG400) and mixturesthereof. The topical formulations may desirably include a compound whichenhances absorption or penetration of the active ingredient through theskin or other affected areas. Examples of such dermal penetrationenhancers include dimethylsulfoxide and related analogs.

The oily phase of the emulsions of this invention is constituted fromknown ingredients in a known manner. While this phase may comprisemerely an emulsifier (otherwise known as an emulgent), it desirablycomprises a mixture of at least one emulsifier with a fat or an oil orwith both a fat and an oil. Optionally, a hydrophilic emulsifier isincluded together with a lipophilic emulsifier which acts as astabilizer. It is also preferred to include both an oil and a fat.Together, the emulsifier(s) with or without stabilizer(s) make up theso-called emulsifying wax, and the wax together with the oil and fatmake up the so-called emulsifying ointment base which forms the oilydispersed phase of the cream formulations.

The choice of suitable oils or fats for the formulation is based onachieving the desired cosmetic properties. Thus the cream shouldoptionally be a non-greasy, non-staining and washable product withsuitable consistency to avoid leakage from tubes or other containers.Straight or branched chain, mono- or dibasic alkyl esters such asdi-isoadipate, isocetyl stearate, propylene glycol diester of coconutfatty acids, isopropyl myristate, decyl oleate, isopropyl palmitate,butyl stearate, 2-ethylhexyl palmitate or a blend of branched chainesters known as Crodamol CAP may be used, the last three being preferredesters. These may be used alone or in combination depending on theproperties required. Alternatively, high melting point lipids such aswhite soft paraffin and/or liquid paraffin or other mineral oils can beused.

Formulations suitable for topical administration to the eye also includeeye drops wherein the active ingredient is dissolved or suspended in asuitable carrier, especially an aqueous solvent for the activeingredient. The active ingredient is optionally present in suchformulations in a concentration of 0.5 to 20%, advantageously 0.5 to 10%particularly about 1.5% w/w.

Formulations suitable for topical administration in the mouth includelozenges comprising the active ingredient in a flavored basis, usuallysucrose and acacia or tragacanth; pastilles comprising the activeingredient in an inert basis such as gelatin and glycerin, or sucroseand acacia; and mouthwashes comprising the active ingredient in asuitable liquid carrier.

Formulations for rectal administration may be presented as a suppositorywith a suitable base comprising for example cocoa butter or asalicylate. Formulations suitable for nasal administration wherein thecarrier is a solid include a coarse powder having a particle size forexample in the range 20 to 500 microns (including particle sizes in arange between 20 and 500 microns in increments of 5 microns such as 30microns, 35 microns, etc), which is administered by aerosol or powderinhalers, of which numerous examples are available. Suitableformulations wherein the carrier is a liquid, for administration as forexample a nasal spray or as nasal drops, include aqueous or oilysolutions of the active ingredient.

Formulations suitable for vaginal administration may be presented aspessaries, tampons, creams, gels, pastes, foams or spray formulationscontaining in addition to the active ingredient such carriers as areknown in the art to be appropriate.

Formulations suitable for parenteral administration include aqueous andnon-aqueous sterile injection solutions which may contain anti-oxidants,buffers, bacteriostats and solutes which render the formulation isotonicwith the blood of the intended recipient; and aqueous and non-aqueoussterile suspensions which may include suspending agents and thickeningagents. The formulations are presented in unit-dose or multi-dosecontainers, for example sealed ampoules and vials, and may be stored ina freeze-dried (lyophilized) condition requiring only the addition ofthe sterile liquid carrier, for example water for injections,immediately prior to use. Extemporaneous injection solutions andsuspensions may be prepared from sterile powders, granules and tabletsof the kind previously described.

The compound of this invention optionally is formulated into controlledrelease compositions in which the release of the compound is controlledand regulated to allow less frequency dosing or to improve thepharmacokinetic or toxicity profile of the invention compound.Controlled release compositions are prepared in accord with knownmethods, many of which involve formulating the active compound with oneor more polymer carriers such a polyester, polyamino acid, polyvinylpyrrolidone, ethylene-vinyl acetate copolymer, methylcellulose,carboxymethylcellulose and/or protamine sulfate. The rate of drugrelease and duration of action optionally is controlled by incorporatingthe active ingredient into particles, e.g. microcapsules, of a polymericsubstance such as hydrogels, polylactic acid, hydroxymethylcellulose,polymethyl methacrylate and the other above-described polymers. Alsosuitable are colloid drug delivery systems such as liposomes,microspheres, microemulsions, nanoparticles, nanocapsules and so on.Depending on the route of administration, the pharmaceuticalcomposition, e.g., tablets, may require protective coatings.

The invention will be more fully appreciated by reference to thefollowing examples, which are to be considered merely illustrative andnot limiting the scope of the invention as claimed.

EXAMPLE 1a Synthesis of5-({6-[2,4-bis(trifluoromethyl)phenyl]pyridazin-3-yl}methyl)-2-(2-fluorophenyl)-5H-imidazo[4,5-c]pyridine

In this method, dimethoxyethane or its related solvents, all having thegeneral formula R¹OR²O(R⁴O)_(a)R³ wherein each of R¹, R², R³ and R⁴ areindependently selected from C1-C6 alkyl and a is 0 or 1, have been foundto be particularly advantageous over the conventional solvent DMF.Typically, each of R¹, R², R³ and R⁴ are independently C₁-C₂ alkyl andusually a is 0. C₁-C₆ alkyl includes fully saturated primary, secondaryor tertiary hydrocarbon groups with 1 to 6 carbon atoms and therebyincludes, but is not limited to methyl, ethyl, propyl, butyl, etc.

Step 1

Compound MW Amount mmoles Equivalents SM 128.56 5 g 38.9 1 TCCA 232.413.62 g 15.6 0.4 CHCl₃ 130 ml

To a solution of the commercially available starting material (SM) inCHCl₃, trichloroisocyanuric acid (TCCA) was added at 60° C. Then thesolution was stirred for 1.5 hrs., cooled down and filtered withHiFlo-Celite. The filtrate was concentrated and dried with vacuum. Theyield was 5.037 g.

Step 2

Compound MW Amount mmoles Equivalents S.M. 163 5.073 g 31.12 1 Core213.2 6.635 g 31.12 1 NaOH (10%) 40 1.245 g 31.12 1 DMF 320 ml

To a solution of core (obtained as described in literature in DMF(dimethylformamide), NaOH was added. Then SM for this step (obtainedfrom step 1) was dissolved in DMF (20 ml) and added to the solutionslowly. The reaction was stirred for 3 hrs, was diluted with water andextracted with EtOAc. The organic layer was dried with Na₂SO₄. Thesolvent was removed and the product recrystallized with DCM(dichloromethane). The yield was 5.7 g.

Step 3

Compound MW Amount Moles Equivalents A 453.79 95 mg 0.209 1 DME 500 ul2N aq. Na₂CO₃ 313 ul 0.626 3 2,4-bisCF₃- 257.93 80.9 mg 0.313 1.5phenylboronic acid Pd(PPh₃)₄ 1155 12 mg 0.0104 0.05

Compound A was dissolved in dimethoxyethane (DME). To this solution wasadded 2,4-bis(trifluromethyl)phenylboronic acid and a 2N aq. Na₂CO₃solution. To the resulting biphasic mixture was added Pd(PPh₃)₄ and thereaction was then heated at 80° C. for 72 hrs. The reaction was cooledto room temperature and filtered through Celite and the Celite washedwith EtOAc. The filtrate was concentrated in vacuo. The residue waspurified on 6 g SiO2 using MeOH/CH2Cl2 to elute compound. The compoundthus obtained was contaminated with PPh₃(O). The product was repurifiedon a 1 mm Chromatotron plate with 0 to 5% MeOH/CH₂Cl₂ in 1% steps. Thepure fractions were combined and concentrated in vacuo, then dried onhigh vacuum for 12 hrs. 11.8 mg of the free base of compound (1) wasobtained with no PPh₃ contamination.

¹H NMR (300 MHz, CD₃OD)

6.20 (s, 2)7.32 (m, 3)7.52 (m, 1)7.78 (d, 1)7.89 (d, 1)7.95 (s, 2)8.15 (m, 3)8.35 (d, 1)9.12 (s, 1)

LC/MS M+H=518 EXAMPLE 1b Synthesis of5-({6-[2,4-bis(trifluoromethyl)phenyl]pyridazin-3-yl}methyl)-2-(2-fluorophenyl)-5H-imidazo[4,5-c]pyridine

This example is directed to an additional method for making compound(1), employing the following schemes.

Methanesulfonic acid was added to 2-fluorobenzoic acid in a reactor withactive cooling keeping T≦50° C. 3,4-Diaminopyridine was then addedportionwise to this cooled slurry, keeping T≦35° C. The contents of thereactor were then heated to 50° C. Phosphorus pentoxide was added in asingle charge. The reaction was then heated at 90-110° C. for at least 3hours. The reaction was sampled for completion by HPLC analysis. Thereaction was cooled to ambient temperature and water was addedportionwise slowly to quench the reaction. The reaction was then dilutedwith water. In solubles were removed by filtration. The pH of thefiltrate was adjusted to 5.5-5.8 with ammonium hydroxide. The reactionwas allowed to self-seed and granulate for ˜4 hours at ambienttemperature. The pH was then adjusted to 8.0-9.3 with ammoniumhydroxide. The slurry was held at ambient temperature for at least 2hours. The solids were isolated by filtration and washed with water,followed by IPE. The wet cake was dried in vacuo at not more than 60° C.until ≦1% water remains. The dry product is core (2).

Summary of Materials M.W. Wt. Ratio Mole ratio 3,4-Diaminopyridine109.13 1.0 1.0 2-Fluorobenzoic acid 140.11 1.4 1.1 Methanesulfonic acid96.1 7.0 8.0 Phosphorus pentoxide 141.94 1.3 1.0 Water 18.02 40 —Isopropyl ether 102.17 5.0 — Ammonium hydroxide 35.09 ~10 —

A solution of compound (2a) in 1,2-dichloroethane was heated to 40-45°C. Trichloroisocyanuric acid was added and the mixture was heated at60-70° C. for at least 2 hours. The reaction was sampled for completionby HPLC analysis. The reaction was cooled to ambient temperature. Celitewas added to absorb insolubles, then solids were removed by filtration.The filtrate was washed with 0.5 N sodium hydroxide solution. Theorganic layer was concentrated to lowest stirrable volume and displacedwith DMF. Core (2) and 10% aqueous sodium hydroxide solution were added.The reaction was stirred at ambient temperature for at least 8 hours.The reaction was sampled for completion by HPLC analysis. An additional10% charge of 10% sodium hydroxide solution was added to the reaction.The reaction was then charged into water to isolate the crude product.After granulating for at least 1 hour, the solids were isolated andwashed with water and isopropyl ether. Ethyl acetate was added andrefluxed (internal T=70-77° C.) for 1-5 hours to dissolve product, thencooled to 18-23° C. slowly over 4-8 hours. The reactor contents wereagitated at 18-23 ° C. for 8-20 hours and solids collected by filtrationand rinsed with ethyl acetate. Low melt (i.e., DSC about 220 degrees C.)amorphous compound (1) was discharged. Amorphous compound (1) wasdissolved in ethyl acetate by heating at reflux (internal T=70-77° C.)for 1-5 hours. Water content is controlled to about 0.2% byazeotropically removing water (with ethyl acetate the upper limit onwater content is about 0.6% by weight; at about 0.9% by weight water theamorphous material will reprecipitate and crystals will not beobtained). The reactor contents are cooled slowly to 18-23° C. over 4-8hours, then agitated at 18-23° C. for 8-20 hours and solids collected byfiltration. The solids were rinsed with ethyl acetate and dried in vacuoat not more than 60° C. to obtain the dry crystalline compound (1).

Summary of Materials M.W. Wt. Ratio Mole ratio3-chloro-6-methylpyridazine 128.56 1.0 1.02,4bis(trifluromethyl)phenylboronic 257.93 4.0 2.0 acid X-Phos 476.720.18 0.05 Palladium acetate 224.49 0.04 0.025 1,2-Dimethoxyethane 90.1216.7 — Potassium carbonate 138.21 2.15 2.0 Water 18.02 7.8 — Copperiodide 190.45 0.037 0.025 Celite — 0.25 — Heptane 100.2 22.4 —

Nuclear Magnetic Resonance (¹H-, ¹³C-, and ¹⁹F-NMR) Spectra

Nuclear magnetic resonance (NMR) spectra of compound (1) is consistentwith the proposed structure. The ¹³C, ¹⁹F, and ¹H-NMR spectra ofcompound (1) in DMSO-d₆ were measured using a Varian Unitylnova-400FT-NMR spectrometer. Spectra are shown in the table below. The NMRchemical shift assignments were established using 2D correlationexperiments (COSY, HSQC, HMBC and HSQCTOCSY).

¹H and ¹³C-NMR chemical shift assignments for Compound (1) referencestandard

At- δC/ppm δF/ppm δH/ppm om (DMSO-d₆) (DMSO-d₆) (DMSO-d₆) 1A 140.16 2A128.32 (q^(a), J_(CF) = 32 Hz)  3A 123.61, m 8.24 (m, 1 H) 4A 130.27 (q,J_(CF) = 34 Hz)  5A 129.54 (q, J_(CF) = 3 Hz)  8.22 (m, 1 H) 6A 133.367.88 (m, 1 H) 7A 123.20 (q, J_(CF) = 273 Hz) −56.4^(b) 8A 123.02 (q,J_(CF) = 275 Hz) −62.0^(b) 1B 158.76 2B 128.16 8.01 (d, 1 H, J = 8.4 Hz)3B 126.20 7.95 (d, 1 H, J = 8.8 Hz) 4B 157.70 5B  60.49 6.17 (s, 2 H) 2C131.86 8.31 (m, 1 H) 3C 112.63 7.86 (m, 1 H) 4C 155.44 6C 168.11 (d,J_(CF) = 6 Hz)  8C 145.08 9C 133.06 9.25 (s, 1 H) 1D 123.11 (d, J_(CF) =10 Hz)  2D 160.46 (d, J_(CF) = 254 Hz) −111.7 3D 116.59 (d, J_(CF) = 22Hz)  7.29 (m, 1 H) 4D 130.84 (d, J_(CF) = 8 Hz)  7.46 (m, 1 H) 5D 124.13(d, J_(CF) = 4 Hz)  7.31 (m, 1 H) 6D 131.72 (d, J_(CF) = 2 Hz)  8.35 (m,1 H) ^(a)multiplicity, s: singlet, d: doublet, q: quartet, m: multiplet^(b)interchangeable signals

Differential Scanning Calorimetry

Compound (1) samples made according to the methods of examples 1a(Research lot 6)) and 1b (remaining samples) were subjected tomeasurement using a Differential Scanning Calorimetry (DSC) apparatus(DSC2010, manufactured by TA Instruments Corporation), under nitrogenatmosphere, sample weight 5±1 mg, temperature rise rate: either 1° C.per min, 5° C. per min or 10° C. per min, open aluminum pan, and indiumstandard as a reference. The enthalpy, extrapolated onset temperatureand apex temperature at an endothermic peak on the obtained DSC curvewere determined.

The DSC results for representative Compound (1) batches are summarizedin Table 1. When the crystal form of Compound (1) produced by theexample lb method was subjected to DSC scan at 1° C./min, the enthalpyof the endothermic peak is about 81 J/g±1 J/g, and the extrapolatedonset temperature is 233.2° C.±2.0 The apex of the endothermic peak is233.9° C.±3.0° C.

TABLE 1 Example DSC values obtained for Compound (1) batches 10° C./minscan 1° C./min scan peak onset main peak peak onset main peak Enthalpy(J/g) Compound (1)Ref Std 235.8 237.2 233.7 234.6 89.5 Compound (1)-A-1n/a n/a 234.8 234.0 — Compound (1)-B-1 Crop 1 235.2 237.4 231.6 232.278.5 Compound (1)-B-1 Crop 2 236.1 238.5 234.3 235.6 80.9 **Research Lot6 220.2 221.3 pending pending 39.1 Note: All ° C. excecpt for enthalpy**5° C./min scan reported for Lot 6

X-Ray Powder Diffractometry

Samples made by methods 1a and 1b were analyzed in the as receivedcondition, only mixing with a spatula prior to analysis. A sample wasfixed to an aluminum cell, and the measurement was performed using anX-ray powder diffractometer (XRD-6000, Shimadzu Lab X, manufactured byShimadzu Corporation, X-ray source: Cu—Kα1 ray, tube voltage: 35 kV,tube electric current: 40 mA, scan speed: 2° per min, continuous scanmode, sampling pitch: 0.02°, scan range: 4-35°, β axis rotation: 60rpm).

Non-micronized, ascicular compound (1) crystals obtained by the example1b method have an X-ray powder diffraction pattern having characteristicdiffraction peaks at diffraction angles 2θ (°) of 13.46, 15.59, 16.90,17.48, 23.05 and 30.15 as measured by X-ray powder diffractometer (FIG.1). Note that the non-micronized “high melt” 235° C. melt ascicularcrystal form of compound (1) tested in this example shows some effectsdue to preferred orientation and particle size. As a result, FIG. 1should be considered merely exemplary because varying the crystal sizeand orientation will change the magnitude of the peaks in the plot.Additionally, the diffraction peak value at the above mentioneddiffraction angle 2θ (°) may show slight measurement error due to themeasurement instrument or measurement conditions and the like.Typically, the measurement error generally is within the range of about±0.3. The specification for the Shimadzu XRD-6000 is ±0.04. In addition,some variation in peak positions can be expected due to product andexperimental variation, so they must be considered approximate.

The 220° C. “low melt” solid state form of compound (1) comprised byproduct made according to the example 1a method (or in the method 1bprior to the reslurry step) gives an X-ray powder diffraction patternconsistent with amorphous material (FIG. 2).

The product of this method 1b is crystalline compound (1) substantiallyfree of amorphous compound. It exhibits an endothermic onset at about235° C. in differential scanning calorimetry (DSC) profile. It exhibitsan approximate heat of fusion (DH_(f)) of 81 J/g (42 KJ/mole)±1 J/g.Crystalline compound (1) is produced substantially free of amorphouscompound (1) by reslurring the reaction product in substantiallyanhydrous crystallization solvent, as described above. Thecrystallization solvent is any solvent or cosolvent mixture in whichcompound (1) will dissolve. Suitable solvents include isopropylacetate/ethyl acetate cosolvent, or ethyl acetate alone.

Substantially anhydrous solvent is defined as solvent containing asufficiently small amount of water that the product compound (1)composition according to method lb contains crystalline compound (1) andless than about 40%, ordinarily less than about 30, 20, 10, 5, 3, 2 or1% by weight of any other form of compound (1) (including amorphouscompound (1)) in the total of all forms of compound (1) in the productcomposition.

In general, substantially anhydrous solvent will contain less than about0.5% -0.6% by weight of the crystallization solvent as water, althoughthe amount of permitted water will vary based on the objectives of theprocess. For example, more water can be present if the desired productis permitted to contain the greater proportions of amorphous compound(1). The determination and selection of the amount of permitted water isentirely within the skill of the artisan and will depend upon a numberof factors, including the nature and identity of the solvent, thepresence of agents for scavenging water, the temperature of the reactionand other conditions.

Compound (1) by example lb typically exhibits intrinsic solubility of0.7 micrograms/ml, a pKa of 5.8, log P of 2.8; and geometric mean (3lots) pH solubility profile at pH 2 of 458 micrograms/ml and at pH 7.3,0.7 micrograms/ml. Geometric mean solubility (3 lots) in simulatedintestinal fluids (fasted: pH 6.4, 0.75 mM lecithin, 3 mM sodiumtaurocholate, 270 mOsmol; fed: pH 5.0, 3.75 mM lecithin, 15 mM sodiumtaurocholate, 635 mOsmol) were 19.1 micrograms/ml (fasted) and 122micrograms/ml (fed).

Measured parameters vary from lot to lot, so all of the foregoingparameters except molecular weight should be considered to beapproximate.

Titration with acids revealed higher solubility with mesylate (>20mg/ml) compared to the chloride (about 0.6 mg/mL) or sulfate (about 0.5mg/mL) counterions.

EXAMPLE 2 Formulation of Compound (1)

Compound (1) formulations are made on a weight by weight basis toachieve 10% w/w active. To make 12 kg solution, exemplary quantitativecompositions of compound (1) capsules, 20 mg and 40 mg are listed below.

Quantitative composition of Compound (1) capsules, 20 mg and 40 mg

Capsule Unit Formula (mg/unit) Compendial Components % w/w 20 mg 40 mgReference Function Compound 1 10.00 20.0 40.0 None Active ingredientOleic Acid 84.55 169.1 338.2 NF Solvent Polysorbate 80 5.00 10.0 20.0 NFSurfactant Butylated Hydroxytoluene 0.10 0.2 0.4 NF Antioxidant (BHT)Butylated Hydroxyanisole 0.35 0.7 1.4 NF Antioxidant (BHA) CapsuleSealing Solution^(a) Capsule sealant Ethanol —^(b) —^(b) —^(b) USP —Purified water —^(b) —^(b) —^(b) USP — Capsule Shell, Size 0 Licaps ™N/A 1 each 1 each None Capsule shell White Opaque Total 100.00 200.0400.0 ^(a)Composition is 1:1 w/w ethanol:water solution. ^(b)Removedduring the capsule sealing process.

-   -   Container/vessel: 12 kg stainless steel    -   Weigh the following in order:    -   0.012 kg butylated hydroxytoluene (0.10%)    -   0.035 kg butylated hydroxyanisole (0.35%)    -   1.2 kg Compound (1) free base (10%).    -   0.6 kg Polysorbate 80 (5%) weighed    -   10.153 kg oleic Acid (equivalent to 84.55 g (84.55%))

Compound (1) capsules, 20 mg or 40 mg, are manufactured through a seriesof unit process steps. Compound (1) drug substance, oleic acid,polysorbate 80, butylated hydroxytoluene (BHT), and butylatedhydroxyanisole (BHA) are mixed until a solution is achieved. Thesolution is filled into 2-piece hard gelatin capsules. Closed capsulesare subsequently sealed with a hydroalcoholic solution, which isevaporated during the sealing process. A vacuum leak test is performedon sealed capsules prior to packaging.

Alternative Formulations

The compound of formula (1) optionally is formulated into a solubilizedform with the following agents:

-   -   Fatty acids (short, medium, and long chained as well as        saturated and unsaturated), typically C4 to C22. Typical fatty        acids are lauric acid, capric acid or oleic acid.    -   Alcohols such as ethanol, benzyl alcohol, glycerol, polyethylene        glycol 200, polyethylene glycol 300, polyethylene glycol 400.    -   Surfactants, including both ionic and non-ionic surfactants.        Examples of non-ionic surfactants are fatty acid esters of        polyoxyethylene sorbitan, sorbitan fatty acid ester,        polyoxyethylene castor oil derivatives, polyoxyethleneglycerol        oxystearate, polyethyleneglycol 60, hydrogenated castor oil,        and/or block copolymers of ethylene oxide and propylene oxide.    -   Antioxidants, for example butylated hydroxyanisole (BHA),        butylated hydroxytoluene (BHT), vitamin E, and/or vitamin E PEG        1000 succinate for chemical stability.    -   Viscosity inducer (silicon dioxide, polyethylene glycols,        titanium oxide and the like).    -   And mixtures of the above

Encapsulation can be performed in a soft elastic gelatin or a hardgelatin or a hard hydroxypropyl methyl cellulose capsule. The liquidformulation (solution or encapsulated solution) provides improved oralbioavailability.

Capsule Filling

The composition and preparation of the soft elastic gelatin capsule iswell known in the art. The composition typically comprises from 30-50%by weight gelatin, 10-40% plasticizer or a blend of plasticizers andabout 25-40% by weight water. Plasticizers can be glycerin, sorbitol orsorbitol derivatives, propylene glycol and the like or a combinationthereof.

Various methods can be used for manufacturing and filling the softelastic gelatin capsules such as rotary, liner or accogel machine andthe like. Hard gelatin or HPMC capsules can be purchased from. Capsugel,Greenwood, S.C. and other suppliers. Capsules are filled manually or bycapsule filling machine.

Formulation Preparation

In general, the compositions of this invention can be prepared in thefollowing manner. The ingredients are mixed in an appropriate vesselsize using an overhead mixer (The mixing tank may be purged withnitrogen). The pharmaceutically acceptable fatty acid and thepharmaceutically acceptable antioxidant are mixed at room temperature.(The solution may be warmed to appropriate temperature if needed, forexample to about 45 degrees C. in the case of lauric acid, in order toliquefy the fatty acid). The compound of formula (1) is added andstirred until dissolved. The pharmaceutically acceptable surfactant isadded with mixing. The appropriate weight of the resulting mixture isfilled into hard gelatin capsules

Additional Formulation Compositions

Formula (1) compound 8.0 PEG 400 82.8 EtOH 9.2 Total 100.0 Formula (1)compound 8.0 EtOH 11.0 PG 7.4 Maisine 35-1 36.8 Cremophor RH40 36.8Total 100.0 Formula (1) compound 8.0 Oleic Acid 92.0 Total 100.0 Formula(1) compound 8.0 Oleic Acid 73.6 EtOH 9.2 Tween 20 9.2 Total 100.0Formula (1) compound 8.00% Oleic Acid 87.40% Tween 80 4.60% Total100.00% FORMULA (1) COMPOUND 20.00% Oleic Acid 80.0% Total 100.0%FORMULA (1) COMPOUND 20.00% Oleic Acid 76.00% Tween 80 4.00% Total100.00% FORMULA (1) COMPOUND 8.00 Oleic Acid 86.47% Tween 80 4.60%Aerosil 200 0.92% BHT 0.01% Total 100.0% FORMULA (1) COMPOUND 8.00 OleicAcid 85.55% Tween 80 4.60% Aerosil 200 1.84% BHT 0.01% Total 100.0%FORMULA (1) COMPOUND 8.00 Oleic Acid 85.55% Tween 80 4.60% Aerosil 2001.84% BHT 0.01% Total 100.0% FORMULA (1) COMPOUND 10.00 Oleic Acid84.55% Tween 80 5.00% BHA 0.35% BHT 0.1% Total 100.0%

EXAMPLE 2a Micronized Formulation of Compound (1)

Micronized drug substance (Jet mill-00 at 60-80 psi; 3-4 microns averagesize, about 7-8 sq. meters/g) was dry blended with lactose,microcrystalline cellulose, croscarmellose sodium, sodium laurylsulfate, tartaric acid, and hydroxypropyl cellulose. The blend wasgranulated by spraying the blend solution. The granules were dried in afluid-bed. The dried granules were sized by passing through a mill, andthen blended with additional microcrystalline cellulose andcroscarmellose sodium. The powder blend was lubricated by addingmagnesium stearate and then compressed into tablets using a rotarytablet press. The tablets were subsequently film-coated.

The table below is a summary of various formulations tested in dogsdosed at 40 mg compound (1), corresponding to approximately 4 mg/kg. Thetable illustrates the superior performance of the solubilized compound(1) formulations.

In-vivo Data Summary Drug Dosage Load Cmax AUC₂₄ F RSD Form ProcessFormula (%) (μM) (μM hr) (%) (%) Solid Powder PIC 50 0.7 2.9 8 52Fill^(a) Solubilized Liquid Capric 20 4.8 25 79 17 Fill acid Lauric 202.6 14.3 44 29 acid Oleic Acid 8 3.8 23 67 27 20 2.1 14 44 56 25 7.9 42125 24 Solid High SLS only 20 0.4 4.4 13 85 Shear^(a) SLS & 20 0.4 2.7 882 Tartaric SLS & 20 0.9 6.9 20 67 Tartaric^(b) Fluid SLS & 20 0.3 4.414 77 bed^(a) Tartaric ^(a)Utilizes micronized API ^(b)Dosed in dogstreated with pentagastrin to reduce stomach pH

EXAMPLE 3 Antiviral Activity of Compound (1)

The compound of this invention exhibits anti-HCV replicon activity(assay described in WO 05/063744) against both genotypes 1a and 1b,extremely low cytotoxicity (>50,000 nM in Huh-7, HepG2 and MT4 cells),and a highly favorable selectivity index. The compound is substantiallyless active against genotype 2a.

Activity of Compound 1 Against HCV Genotype 1b and 1a Replicons

HCV genotype 1b (Con-1/lucneo) and 1a (H77/neo) replicon cells wereincubated with serial dilutions of compound (1) 2′C-methyl adenosine(2′CMeA) or IFNα for 3 days in the absence or presence of 40 mg/mL humanserum albumin (HSA). After incubation, replicon RNA levels in thetreated cells were determined by either a luciferase reporter assay (1breplicon) or a quantitative real-time PCR assay (1a replicon) and thedata points were used to calculate EC₅₀ (50% effective inhibitingconcentration) values for the inhibitors. Compound (1) was shown toinhibit both genotype 1b and genotype 1a replicons with EC₅₀ values of0.6 and 3.6 nM, respectively (Table A). In the presence of human serumalbumin, the EC₅₀ value of Compound (1) was increased to 11 nM.

TABLE A Activity of Compound (1) against HCV Genotypes 1a and 1bReplicons EC₅₀ [nM]^(a) HCV 1b-lucneo 40 Compound HCV 1b-lucneo mg/mLHSA HCV-1a 1 0.6 ± 0.28  11 3.6 ± 1.4 2′CMeA 175 ± 70  250 170 IFN-α 2IU/mL n.d. n.d. n.d., not determined; HSA, human serum albumin ^(a)MeanEC₅₀ value and standard error determined from at least 4 independentexperiments

Activity of Compound (1) Against HCV Genotype 1a Replicon and Virus

The antiviral activity of compound (1) against HCV genotype 2a wastested in cells chronically infected with the genotype 2a virus as wellas in cells replicating a subgenomic 2a replicon. Huh-7 cells containingchronically replicating HCV genotype 2a (J6/JFH-Rluc) virus orsubgenomic replicons were cultured with compound (1) or 2′CMeA for 3days in the absence of human serum albumin. After cultivation, theamount of luciferase in 2a-virus containing cells and HCV NS3 proteaseactivity in the 2a replicon-containing cells was determined usingPromega's luciferase assay and a novel time-resolved fluorescence assay,respectively.

The antiviral activity of compound (1) was significantly reduced in boththe HCV-2a chronically infected cell culture model (EC₅₀=2.9 μM) and the2a subgenomic replicon model (EC₅₀=21.9 μM) compared to Huh-7 cellsreplicating an HCV-1b subgenomic replicon (EC₅₀=0.0006 μM) (Table 2).Taken together, these results suggest that the reduction in potency forcompound (1) against HCV genotype 2a may be due to the genotypicdifferences between genotype 1 and genotype 2 of HCV.

TABLE B Activity of Compound (1) against HCV Genotypes 1b and 2a EC₅₀[nM]^(a) HCV 1b-lucneo HCV 2a (subgenomic (subgenomic HCV-2a Compoundreplicon) replicon) (reporter virus) 1 0.6 ± 0.28 21898 ± 18972 2900 ±1250 2′CMeA 175 ± 70  1610 ± 1099 194 ± 26  IFN-α 2 IU/mL n.d. 1.2 IU/mLn.d., not determined; HSA, human serum albumin ^(a)Mean EC₅₀ value andstandard error determined from at least 4 independent experiments

Compound (1) was evaluated for its cytotoxicity in a variety of celltypes including HCV replicon-containing cell lines (Huh-7, SL3 and MH4)and non-replicon-containing cell lines (HepG2, MT4), using aCellTiter-Glo Luminescence Cell Viability assay (Promega). No toxiceffects were observed in any of the cell lines at the highestconcentration tested (50 μM) (Table C). These results, coupled with itspotent antiviral activity (EC₅₀=0.62-3.6 nM) in HCV-1b and HCV-1areplicons, indicates a high selectivity index (CC₅₀/EC₅₀>13,000-80,000)for compound (1).

TABLE C Cytotoxicity of compound (1) in HCV Replicon Containing CellLines CC₅₀ [μM]^(a) Huh-7 Compound lucneo^(b) SL3^(b) MH4^(b) HepG2 MT41 >50 >50 >50 >50 >50 2′CMeA 7.2 ± 6 3.9 16 24.3 ± 2.1 3.5 ± 1.9 n.d.,not determined; HSA, human serum albumin ^(a)Mean CC₅₀ value andstandard error determined from at least 4 independent experiments^(b)HCV replicon-containing cell lines

Anti-HCV Activity of Compound (1) in Combination with IFN In Vitro

Pegylated interteron-α (PEG-IFN-α), in combination with ribavirin,represents the current standard of care for HCV-infected patients. Invitro combination studies of compound (1) and IFN-α were performed inreplicon cells. Data was analyzed using the MacSynergy templatedeveloped by Prichard and Shipman. Results from these studies suggest anadditive interaction between compound (1) and IFN-α.

EXAMPLE 4 Antiviral, Pharmacokinetic and Safety Data for Compound (1) ina Phase-1, First-In-Human Trial in HCV Genotype 1-Infected Subjects

A randomized, double-blind, placebo controlled trial was designed toevaluate the safety/tolerability, phamacokinetics and antiviral activityof single (in Part A) and multiple (in Part B) doses of Compound (1)(oleic acid solution, above) in subjects chronically infected with HCVgenotype 1 (GT-1) without decompensated cirrhosis. Prospective subjectsare 18-60 years of age, are HCV treatment naïve, and are in general goodhealth.

In completed Part A, five successive cohorts of 6 subjects wererandomized (5:1) to receive single ascending doses of Compound 1 (40,120, 240, 240-with food, or 480 mg) or placebo. In ongoing Part B, foursuccessive cohorts of 12 subjects are randomized (10:2) to receivemultiple ascending doses of Compound 1 (40 mg BID, 120 mg BID, 240 mgQD, 240 mg BID) or placebo, over 8 days.

Thirty-one subjects enrolled in Part A were of mean age 43.6 years,predominantly male (20/31), Caucasian (25/31), and infected with eitherHCV Genotype-1a (24) or 1b (6). Median (range) baseline HCV viral loadwas 6.6 Log¹⁰ RNA IU/mL (5.2-7.3). Single doses of compound (1) werewell tolerated, with no serious or treatment-limiting adverse events(AEs) reported. The most common AE was headache. All AEs were mild inseverity, with the exception of one moderate headache. There were noGrade 3 or 4 treatment emergent laboratory abnormalities.

Median compound (1) plasma half-life ranged from 10 to 15 hours acrosscohorts. Systemic exposure was increased approximately 2-fold whencompound (1) was administered with a high fat meal. Mean compound (1)concentration 24 hours after the 240 mg fasted dose dosing was ˜7-foldhigher than the protein binding adjusted in vitro HCV GT-1b RepliconEC₅₀ value. Following single-dose exposure, maximal antiviral effect wasobserved at 24 hours, with median declines ranging from 0.46 to 1.49Log¹⁰ HCV RNA IU/mL across cohorts. Individual HCV RNA declines amongall compound (1) recipients ranged from 0.19 to 2.54 log¹⁰ IU/mLfollowing single-dose exposure.

This is the first clinical demonstration of antiviral activity ofcompound (1). Single dose exposure to compound (1) was well tolerated,demonstrated favorable PK properties and potent antiviral activity.

EXAMPLE 5

The anti-HCV replicon activity of compound (1) was compared to that of aprior art (WO 05/063744) compound, the compound of formula (4)

Unexpectedly compound (1) was about 330 times more potent than thecompound of formula (4).

1-25. (canceled)
 26. A method for the preparation of compound (1)comprising providing the intermediate (2)

coupling 2,4-bis(trifluoromethyl)phenylboronic acid to3-chloro-6-methylpyridazine to produce compound (2a)

treating compound (2a) with a chlorinating agent to produce thealkylating agent (3)

and using alkylating agent (3) to alkylate intermediate (2) under basicconditions to yield compound (1)


27. A compound of formula (3)

and its analogues substituted with methyl rather than chloromethyl, andits analogues substituted with bromo, fluro or iodo in place of chloro.